The complement protein C5 is a central component of the complement system; a key part of the innate immune system. The complement system is an intricate immune surveillance system with numerous tasks in tightly controlled, diverse processes. It functions as a first line host defense system against infection by other organisms, and also in discriminating healthy host tissues from cellular debris and apoptotic and necrotic cells. Furthermore, it is involved in clearance of immune complexes, regulation of the adaptive immune response, promotion of tissue regeneration, angiogenesis, mobilization of stem cells and development of the central nervous system (Woodruff et al. Mol Immunol 2011, 48 (14):1631-1642); Ricklin et al. Nat Immunol 2010, 11(9):785-795). Any trigger, for example erroneous or unrestricted activation or insufficient regulation, that disturbs the fine balance of complement activation and regulation may lead to pathologic conditions including self-attack of the host's cells leading to extensive tissue damage.
The complement system consists of about 30 proteins. There are three pathways to initiate complement; the classical pathway that employs C1q to recognize immune complexes on the surface of cells; the lectin pathway that is initiated when mannose-binding lectin (MBL) recognizes certain sugars; and the alternative pathway that is initiated spontaneously by hydrolysis of complement factor 3 (C3), a process suppressed by certain mammalian cell surface molecules not present on invading pathogens. The alternative pathway also acts as an amplification loop for the complement system. All three pathways converge at the level of C3. Cleavage of C3 into C3a and C3b leads to the formation of a convertase that in turn cleaves complement factor 5 (C5) into C5a and C5b. C5a is a very potent attractant of various immune cells while C5b oligomerizes with C6-9 to form a pore known as the membrane attack complex (MAC) or sometimes the terminal complement complex (TCC). Activation of the complement system leads to a number of mechanisms with the purpose of neutralizing the pathogen; formation of MAC on the surface of a cell such as an invading bacteria leads to lysis, deposition of C3 and C4 cleavage products C3b and C4b aids opsonization leading to phagocytosis of the pathogen by macrophages and anaphylatoxins such as C3a and C5a attracts monocytes and neutrophils to the site of activation, up-regulates surface markers leading to increased immunologic susceptibility and to the release of cytokines.
C5 is a 190-kDa glycoprotein comprised of 2 disulfide-linked polypeptide chains, alpha and beta, with a molecular mass of 115 and 75 kDa, respectively (Tack et al. Biochem 1979, 18:1490-1497). Haviland et al. (J Immun 1991, 146: 362-368) constructed the complete cDNA sequence of human complement pro-05, which is predicted to encode a 1,676-amino acid pro-molecule that contains an 18-amino acid leader peptide and a 4-amino acid linker separating the beta and alpha chains (SEQ ID NO: 251). Since C5 is common to all pathways of complement activation, blocking C5 will stop the progression of the cascade regardless of the stimuli and thereby prevent the deleterious properties of terminal complement activation while leaving the immunoprotective and immunoregulatory functions of the proximal complement cascade intact.
The complement system's key role in the defense against pathogens in general makes it an interesting target for pharmaceutical intervention. This is emphasized by the fact that many mutations or impaired regulation of complement is involved in various diseases and conditions. These include increased susceptibility to auto-immune diseases such as systemic lupus erythematosis (SLE) where deposition of immune complexes triggers the classical pathway (Manderson et al. Annu Rev Immunol 2004, 22:431-456). In addition, mutations of the complement proteins C1-C5 often result in SLE or SLE like symptoms. Other autoimmune diseases with a strong involvement of the complement system are rheumatoid arthritis (RA) where immune complexes may activate complement in the RA joint, Sjögren's syndrome, dermatomyositis and other autoantibody driven diseases such as Guillain-Barré syndrome (GBS), Fisher syndrome (Kaida et al. J. Neuroimmun 2010, 223:5-12), different types of vasculitis, systemic sclerosis, anti-glomerular basement membrane (anti-GBM) and anti-phospholipid syndrome (APS) (Chen et al. J Autoimmun 2010, 34:J276-J286). Furthermore, complement inhibition have been proven effective in animal models of such different conditions as periodontitis (Abe et al. J Immunol 2012, 189:5442-5448), wound healing (Cazender et al. Clin Dev Immunol 2012, on-line publication), tumor growth (Markiewski et al. Nat Immunol 2008, 9:1225-1235) and diseases of the eye such as uveitis and age-related macular degeneration (AMD) (Copland et al. Clin Exp Immunol 2009, 159:303-314).
Antibodies targeted to human complement C5 are known from, e.g., WO 95/29697; WO 02/30985; and WO 2004/007553. Eculizumab (SOLIRIS) is a humanized monoclonal antibody directed against protein C5 and prevents cleavage of C5 into C5a and C5b. Eculizumab has been shown to be effective in treating paroxysmal nocturnal hemoglobinuria (PNH), a rare and sometimes life threatening disease of the blood characterized by intravascular hemolytic anemia, thrombophilia and bone marrow failure, and is approved for this indication. Eculizumab was also recently approved by the FDA for treatment of atypical hemolytic syndrome (aHUS), a rare but life threatening disease caused by loss of control of the alternative complement pathway leading to over-activation manifested as thrombotic microangiopathy (TMA) leading to constant risk of damage to vital organs such as kidney, heart and the brain. In aHUS, transplantation of the damaged organ only temporarily helps the patient as the liver continues to produce the mutated form of controlling protein (most often complement factor H or other proteins of the alternative pathway). A related disease with a transient acute pathophysiology is HUS caused by infection of Shiga toxin positive E. coli (STEC-HUS) and there are promising clinical data suggesting efficacy also for this condition (Lapeyraque et al, N Engl J Med 2011, 364:2561-2563). Finally, the C5 blocking antibody Eculizumab has proven efficacious in preventing antibody mediated rejection (AMR) in recipients of highly mismatched kidneys (Stegall, M. D. et al. Am J Transplant 2011, 11:2405-2413), and in treating autoimmune neuropathies such as neuromyelitis optica and myasthenia gravis (Pittock et al. Lancet Neurol 2013, 12:554-562; Howard et al. Muscle Nerve 2013, 48:76-84).
Apart from full length antibodies, single-chain variable fragments (scFV), minibodies and aptamers targeting C5 are described in literature. These C5 inhibitors may bind to different sites (epitopes) on the C5 molecule and may have different modes of action. For example, whereas Eculizumab interacts with C5 at some distance of the convertase cleavage site, the minibody MUBODINA interacts with the cleavage site of C5. The C5 inhibitory protein Ornithodoros moubata Complement Inhibitor (OmCI, Nunn, M. A. et al. J Immunol 2005, 174:2084-2091) from soft tick Ornithodoros moubata has been hypothesized to bind to the distal end of the CUB-05d-MG8 superdomain, which is close to the convertase cleavage site (Fredslund et al. Nat Immunol 2008, 9 (7):753-760). In contrast to the three proteins mentioned above inhibiting cleavage of C5, the monoclonal antibody TNX-558 binds to a C5a epitope present both on intact C5 and released C5a without inhibiting the cleavage of C5. (Fung et al. Clin Exp Immunol 2003, 133 (2):160-169).
C5 binding polypeptides, comprising a C5 binding motif, are disclosed in the International Patent Application No. PCT/SE2013/050139, published as WO 2013/126006. In particular, WO 2013/126006 discloses a C5 binding motif, BM, consisting of the amino acid sequence
(SEQ ID NO: 288)EX2X3X4A X6X7EID X11LPNL X16X17X18QW X21AFIX25 X26LX28D,wherein, independently of each other,X2 is selected from H, Q, S, T and V;X3 is selected from I, L, M and V;X4 is selected from A, D, E, H, K, L, N, Q, R, S, T and Y;X6 is selected from N and W;X7 is selected from A, D, E, H, N, Q, R, S and T;X11 is selected from A, E, G, H, K, L, Q, R, S, T and Y;X16 is selected from N and T;X17 is selected from I, L and V;X18 is selected from A, D, E, H, K, N, Q, R, S and T;X21 is selected from I, L and V;X25 is selected from D, E, G, H, N, S and T;X26 is selected from K and S; andX28 is selected from A, D, E, H, N, Q, S, T and Y.
Examples of specific C5 binding motifs, as previously disclosed in WO 2013/126006, are shown as SEQ ID NO: 1-248 in the present patent application.
It is known from WO 2013/126006 that additional peptides or polypeptides may improve stabilization of C5 binding polypeptides. One example of such a polypeptide is the albumin binding domain (ABD) shown as SEQ ID NO: 250 in the present description. Other examples of suitable albumin binding domains are disclosed in WO 2009/016043 and WO 2012/004384. An ABD-extended polypeptide binds to serum albumin in vivo, and benefits from its longer half-life, which increases the net half-life of the polypeptide itself (see e.g. WO 91/01743).
The continued provision of agents with comparable C5 blocking activity remains a matter of substantial interest within the field. In particular, there is a continued need for molecules that prevent the terminal complement cascade as well as the formation of the pro-inflammatory molecule C5a. Of great interest is also a provision of uses of such molecules in the treatment of disease.